Method for analyte examination

ABSTRACT

A method may include steps of binding an analyte to a ligand, exposing the analyte to light of differing wavelengths, and observing a difference in spectral response of the analyte at the differing wavelengths. Further steps may include comparing the observed difference with a known difference in spectral response of a reference substance at the differing wavelengths, and determining whether the observed difference meets a predetermined threshold of identity with the known difference. The method may be performed with reference to a surface having a reflective area with a known size a non-reflective area adjacent to the reflective area. The method may thus include the steps of providing a ligand in alignment with the non-reflective area of the surface, and binding an analyte to the ligand in a position reaching across the reflective area. Further steps may include forming an image of the analyte and the reflective area, observing the size of analyte relative to the reflective area in the image, and comparing the observed size of the analyte with a known size of a reference subject to determine a degree of identity between the analyte and the reference subject.

BACKGROUND

In the prior art, as shown for example in U.S. Pat. No. 8,735,846, a biosensor disk may have a spiral data path encoded with digital data. The data is established by a sequence of pits and lands on the surface of the disk. As the disk is rotated in a reading system, light from a laser or light emitting diode is directed onto the disk at the spiral data path. The system reads the encoded data as indicated by the light reflected from the pits and lands.

The disk further has detector chambers for containing analytes. Ligands are provided to bind specific analytes in place within the chambers. The detector chambers are interposed between the reader system and the spiral data path such that analytes in the chambers can obscure the transmission of light to and from the data path. Accordingly, alterations in the data read by the system indicate the presence of the specific analytes corresponding to the ligands in the chambers where the data path is obscured.

SUMMARY

A method may include steps of binding an analyte to a ligand, exposing the analyte to light of differing wavelengths, and detecting a difference in spectral response of the analyte at the differing wavelengths. Further steps may include comparing the detected difference with a known difference in spectral response of a reference substance at the differing wavelengths, and determining whether the detected difference meets a predetermined threshold of identity with the known difference.

The method may be performed with reference to a surface having a reflective area with a known size and a non-reflective area adjacent to the reflective area. The method may thus include the steps of providing a ligand in alignment with the non-reflective area of the surface, and binding an analyte to the ligand in a position reaching across the reflective area. Further steps may include forming an image of the analyte and the reflective area, observing the size of analyte relative to the reflective area in the image, and comparing the observed size of the analyte with a known size of a reference subject to determine a degree of identity between the analyte and the reference subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an apparatus including a biosensor disk and a system for examining a sample on the disk.

FIG. 2 is a side view of a part of the disk shown in FIG. 1.

FIG. 3 is an exploded sectional view of parts of the disk of FIG. 1.

FIG. 4 is a view taken on line 4-4 of FIG. 3.

FIG. 5 is a side view of another part of the disk of FIG. 1.

FIG. 6 is a view taken on line 6-6 of FIG. 3.

FIG. 7 is a view similar to FIG. 6 showing sample units for examination on the disk.

FIG. 8 is a view similar to FIG. 7 showing an image of a sample unit on the disk.

FIG. 9 is a view similar but opposite to the view of FIG. 8.

FIG. 10 is a view showing an image of another sample unit on the disk.

FIG. 11 is a view similar but opposite to the view of FIG. 10.

FIG. 12 is a view showing an image of yet another sample unit on the disk.

FIG. 13 is a view similar to FIG. 12 but shows an image formed with light of a differing wavelength.

DETAILED DESCRIPTION

The apparatus shown in the drawings has parts that can be used to perform steps that are examples of the steps recited in the method claims. These examples are described here to provide enablement and best mode without imposing limitations that are not recited in the claims.

As shown schematically in FIG. 1, an apparatus includes a biosensor disk 10 and a system 20 for examining a sample on the disk 10. The parts of the disk 10 that are shown in FIG. 1 include a detection substrate 30 and a reflection substrate 32. Discrete units 35 of the sample, which contains biological or other analytes, are distributed within the disk 10 between the detection substrate 30 and the reflection substrate 32. The detection substrate 30 is transparent as needed for the sample units 35, when viewed from that side of the disk 10, to be visible on a background at the reflection substrate 32.

The parts of the system 20 in the example of FIG. 1 include a liquid crystal display (LCD) panel 40, a condenser lens 42, and a sensor array 46. In operation, the disk 10 rotates in its plane above the LCD panel 40. The LCD panel 40 emits light in a band of wavelengths. The condenser lens 42 condenses the emitted light into a spot of greater electromagnetic intensity comprised of the multiple wavelengths in the band, and directs it to the disk 10. Light reflected from the disk 10 reaches the sensor array 46.

As a sample unit 35 on the rotating disk 10 moves across the path of light shown in the drawing, a processor 48 forms one or more images of the sample unit 35 on the background of the reflection substrate 32. By considering parameters such as size, shape and position of the sample unit 35 relative to the background in the images, the processor 48 can correlate those parameters with corresponding parameters of a reference substance, and can thus determine a degree of identity between the sample unit 35 and the reference substance. By comparing images formed at differing wavelengths, the processor 48 can compare the spectral response of the sample unit 35 with a known spectral response of the reference substance at the differing wavelengths, and can thus determine a further degree of identity between the sample unit 35 and the reference substance.

As shown separately in FIG. 2, the detection substrate 30 has an upper side surface 50 which, in the assembled disk 10 of FIG. 1, faces toward the reflection substrate 32. Rows of assay regions 52 extend radially across the upper side surface 50, and are equally spaced apart circumferentially around the upper side surface 50. Ligands 54 are provided at each assay region 52 to hold the sample units 35 in place between the two substrates 30 and 32, as shown in FIG. 3. Specifically, the ligands 54 selected for the imaging process are known to bind, accept or otherwise capture a specific reference substance that the process seeks to detect in the sample units 35. Other features of the disk 10 can be provided as described in U.S. Pat. No. 8,735,846, which is incorporated by reference, including fluidic channels 56 for conveying the sample units 35 to the ligands 54, and a protective substrate 58.

The ligands 54 at each assay region 52 are arranged to provide a pattern of binding and non-binding sites at the assay region 52. As shown partially in FIG. 4, the arrangement of ligands 54 in this particular example provides a uniformly repeating pattern of binding sites 60 shaped as narrow linear areas. The binding sites 60 delineate a corresponding pattern of non-binding sites 62 which, in the given example, are shaped as triangles with sides at the surrounding binding sites 60 and corners where the binding sites 60 intersect. The dimensions of the binding and non-binding sites 60 and 62 are known and stored as baseline data for the imaging process.

As shown separately in FIG. 5, the reflection substrate 32 has a lower side surface 70 that faces the detection substrate 30. The lower side surface 70 also has rows of assay regions 72. Each assay region 72 on the reflection substrate 32 is located directly opposite an assay region 52 on the detection substrate 30. Moreover, each assay region 72 on the reflection substrate 32 has reflective surface areas 74 (FIG. 6) with the same triangular shape, size and pattern of arrangement as the non-binding sites 62 (FIG. 4) on the detection substrate 30. The reflective surface areas 74 are located directly opposite the non-binding sites 62. Each assay region 72 on the lower side surface 70 further has contiguous non-reflective surface areas 76 in an intersecting linear arrangement that matches and faces oppositely toward the linear binding sites 60 on the opposed assay region 52. Like the dimensions of the binding and non-binding sites 60 and 62, the corresponding dimensions of the reflective and non-reflective areas 74 and 76 are known and stored as baseline data for the imaging process.

In operation, the system 20 performs the imaging process in a field of view having the reflection substrate 32 as a background, as noted above. This can begin with a baseline image of the reflection substrate 32 without any sample units 35, as shown in FIG. 6. Such a baseline image may serve as a reference for observing the position, size and/or shape of sample units 35 on the same background in other images.

An image of the reflection substrate 32 with sample units 35 in the foreground may appear as shown partially in FIG. 7. As shown separately in FIG. 8, one such sample unit 35 is located entirely within a reflective surface area 74. That sample unit 35 is likewise located entirely within the opposed non-binding site 62 (FIG. 9). Since the sample unit 35 is shown to have reached that location without being bound by the ligands 54 on the detection substrate 30, its position strongly suggest the absence of the reference substance in this sample unit 35. If this unbound position of the sample unit 35 is taken as a threshold indication of distinction from the reference substance, the sample unit may be dismissed as lacking the reference substance.

In the image shown partially in FIG. 10, a sample unit 35 is shown to overlie a non-reflective surface area 76 on the reflection substrate 32, and to reach across adjacent triangular reflective surface areas 74. This sample unit 35 is thus shown to be bound to the detection substrate 30 at the pattern of binding sites 60 provided by the ligands 54 (FIG. 11), and to reach onto adjacent triangular non-binding sites 62. Such binding of the sample unit 35 to the ligands 54 indicates the presence of the reference substance. Additionally, the shape of the sample unit 35 may be observed directly, and its size may be observed by comparison with the known dimensions of the reflective and non-reflective surface areas 74 and 76. The degree of binding, the shape, and the size of the sample unit 35 can be compared with their known counterparts for the reference substance to assess the similarities or differences indicated for the sample unit 35 and the reference substance. If these comparisons satisfy threshold requirements of similarity, the sample unit 35 may be accepted as including the reference substance.

As an intermediate example, the image shown partially in FIG. 12 includes a sample unit 35 with a lesser degree of binding with the ligands 54, as compared with the example of FIG. 10. This is a lesser indication that the sample unit 35 includes the reference substance. The shape of the sample unit 35 is the same, but the different size provides a correspondingly different comparison with the reference substance. If these observations of bonding, shape and size are considered inconclusive, the imaging process may proceed with spectral response as a parameter.

Specifically, an image of the sample unit 35 is formed with light in a first band of wavelengths. In this example the image of FIG. 12 is taken as the image formed at the first band of wavelengths. The LCD panel 40 can then be operated to emit light in a second band of wavelengths different from the first, and another image is then formed with the light in the second band of wavelengths. This could be an image as shown in FIG. 13. A comparison of the images of FIGS. 12 and 13 shows that the sample unit 35 has greater reflectance transmittance of light in the first band of wavelengths and greater absorption of light in the second band of wavelengths. This difference in spectral response of the sample unit is compared with a known difference in spectral response of the reference substance at the first and second bands of wavelengths. It is then determined whether or not the detected difference in spectral response meets a predetermined threshold of identity with the known difference.

In an alternative example, instead of using differing bands of wavelengths, light of only a single wavelength could be used for any one or more of the multiple exposures performed in a spectral response stage of the method, as long as multiple exposures differ in wavelength sufficiently to obtain a detectable difference in spectral response.

An additional feature of the disk 10 is shown in FIG. 5 where the lower side surface 70 of the reflection substrate 32 has non-reflective regions 80 reaching fully between the rows of assay regions 72. As a non-reflecting region 80 on the rotating disk 10 moves across the path of light shown in FIG. 1, it blocks the transmission of reflected light from the disk 10 to the sensor array 46. This enables the method to include an intervening discharging step between the formation of successive images by effectively exposing the sensor array 46 to darkness so that the sensor elements can fully drain the charges generated in one or more previous exposures to light reflected form the disk 10.

This written description sets for the best mode of carrying out the invention, and describes the invention so as to enable a person skilled in the art to make and use the invention, by presenting examples of elements recited in the claims. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples, which may be available either before or after the application filing date, are intended to be within the scope of the claims if they have elements that do not differ from the literal language of the claims, or if they have equivalent elements with insubstantial differences from the literal language of the claims. 

What is claimed is:
 1. A method comprising: binding an analyte to a ligand; exposing the analyte to light of a first wavelength; exposing the analyte to light of a second wavelength different from the first wavelength; observing a difference in spectral response of the analyte at the first and second wavelengths; comparing the observed difference with a known difference in spectral response of a reference substance at the first and second wavelengths; and determining whether the observed difference meets a predetermined threshold of identity with the known difference.
 2. A method as defined in claim 1 where the binding step binds the analyte to a ligand on a disk, and the exposing steps are performed while rotating the disk.
 3. A method as defined in claim 2 wherein the binding step binds the sample to the disk at a binding site located opposite a non-reflective surface area of the disk.
 4. A method as defined in claim 3 wherein the difference in spectral response of the analyte is observed where the analyte reaches across a reflective surface area of the disk.
 5. A method as defined in claim 4 further comprising the steps of forming an image of the analyte and the reflective surface area, observing the size of analyte relative to the reflective surface area in the image, and comparing the observed size of the analyte with a known size of the reference subject to determine a degree of identity between the analyte and the reference subject.
 6. A method as defined in claim 1 where the exposing steps are performed sequentially.
 7. A method as defined in claim 6 wherein the exposing steps include charging a sensor array with light reflected between a light source and the sensor array, and the method further includes an intervening step of interposing a non-reflective surface between the light source and the sensor array and allowing the sensor array to discharge during the intervening step.
 8. A method as defined in claim 1 wherein the observing step includes the steps of forming an image of the analyte with the light of the first wavelength, forming an image of the analyte with the light of the second wavelength, and observing a difference between the images of the analyte.
 9. A method as defined in claim 1 further comprising the steps of providing a surface having a reflective area with a known size a non-reflective area adjacent to the reflective area, providing the ligand in alignment with the non-reflective area of the surface, and binding the analyte to the ligand in a position reaching from the non-reflective area across the reflective area.
 10. A method as defined in claim 9 further comprising the steps of forming an image of the analyte and the reflective area, observing the size of analyte relative to the reflective area in the image, and comparing the observed size of the analyte with a known size of the reference subject to determine a degree of identity between the analyte and the reference subject.
 11. A method comprising: forming an image of a surface having reflective areas separated by non-reflective areas; binding an analyte to a ligand in a position reaching across the surface from a non-reflective area to a reflective area; forming a first image of the analyte with light of a first wavelength; comparing the first image of the analyte with the image of the surface to identify a reflective surface area covered by the analyte; forming a second image of the analyte with light of a second wavelength different from the first wavelength; observing a difference between the first and second images of the analyte at the covered reflective surface area; comparing the observed difference with a known difference in spectral response of a reference subject at the first and second wavelengths; and determining whether the observed difference meets a predetermined threshold of identity with the known difference.
 12. A method as defined in claim 11 wherein the image forming steps are performed while moving the surface.
 13. A method as defined in claim 11 wherein the image forming steps include reflecting light from the analyte while moving the surface.
 14. A method as defined in claim 11 wherein the image forming steps include transmitting light through the analyte to covered reflective surface area, and reflecting light from the covered reflective surface area through the analyte.
 15. A method as defined in claim 11 where the first and second images are formed sequentially.
 16. A method as defined in claim 15 wherein the step of forming the first image includes charging a sensor array with light reflected between a light source and the sensor array, the step of forming the second image includes charging the sensor array with light reflected between the light source and the sensor array, and the method further includes an intervening step of interposing a non-reflective surface between the source and the sensor array and allowing the sensor array to discharge during the intervening step.
 17. A method as defined in claim 11 further comprising the steps of forming an image of the analyte and the reflective surface area, observing the size of analyte relative to the reflective surface area in the image, and comparing the observed size of the analyte with a known size of the reference subject to determine a degree of identity between the analyte and the reference subject.
 18. A method comprising: providing a surface having a reflective area with a known size a non-reflective area adjacent to the reflective area; providing a ligand in alignment with the non-reflective area of the surface; binding an analyte to the ligand in a position reaching across the reflective area; forming an image of the analyte and the reflective area; observing the size of analyte relative to the reflective area in the image; and comparing the observed size of the analyte with a known size of a reference subject to determine a degree of identity between the analyte and the reference subject.
 19. A method as defined in claim 18 further comprising the steps of observing the shape of the analyte in the image, and comparing the observed shape of the analyte with a known shape of the reference subject to determine a degree of identity between the analyte and the reference subject.
 20. A method as defined in claim 18 wherein the image of the analyte and the reflective area is formed with light of a first wavelength, and further comprising the steps of forming an image of the analyte and the reflective area with light of a second wavelength different from the first wavelength, observing a difference in spectral response of the analyte at the first and second wavelengths, comparing the observed difference with a known difference in spectral response of the reference subject at the first and second wavelengths, and determining whether the observed difference meets a predetermined threshold of identity with the known difference. 